Validation of reference genes for real-time quantitative PCR normalisation in non-heading Chinese cabbage

Non-heading Chinese cabbage is an important vegetable crop that includes pak choi, caixin and several Japanese vegetables like mizuna, mibuna and komatsuna. Gene expression studies are frequently used to unravel the genetics of complex traits and in such studies the proper selection of reference genes for normalisation is crucial. We assessed the expression of 13 candidate reference genes including ACTIN, ACTIN-1, ACTIN-2, GAPDH, Tub_alpha, CyP, EF1-alpha, 18SrRNA, UBQ, UBC30, PPR, PP2A and MDH. Their expression stabilities were analysed using two programs, geNorm and NormFinder, in 20 different samples that represent four strategic groups. Results showed that no single gene was uniformly expressed in all tested samples. ACTIN and CyP are proposed as good reference genes when studying developmental stages. CyP, Tub_alpha and UBC30 are good reference genes when studying different tissues (from flowering to seed set). CyP and Tub_alpha are the most stable reference genes under biotic stress treatments using the fungi Peronospora parasitica and Alternaria brassicicola. UBC30, EF1-alpha and ACTIN are recommended for normalisation in abiotic stress studies, including hormone, salt, drought, cold and heath treatments. Moreover, at least five reference genes (ACTIN, CyP, UBC30, EF1-alpha and UBQ) are required for accurate qRT-PCR data normalisation when studying gene expression across all tested samples.